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ISSN No. (Print): 0975-1130ISSN No. (Online): 2249-3239

PRs proteins and their Mechanism in PlantsFarshid Golshani*, Barat Ali Fakheri*, Elham Behshad** and Roghayeh Mohammadpour Vashvaei***

*Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, IRAN.**Department of Genetics, Faculty of Science, University of Zabol, IRAN.

***Department of Agronomy, Faculty of Agriculture, University of Zabol, IRAN.

(Corresponding author: Farshid Golshani)(Received 10 January, 2015, Accepted 06 February, 2015)

(Published by Research Trend, Website: www.researchtrend.net)

ABSTRACT: Fungi are far more complex organisms than viruses or bacteria and can developed numerousdiseases in plants that cause loss of big portion of the crop every year. Plants have developed variousmechanisms to defend themselves against these fungi which include the production of low molecular weightsecondary metabolites, proteins and peptides having antifungal activity. Pathogenesis-related proteins (PRs)(initially named 'b' proteins) have focused an increasing research interest in view of their possibleinvolvement in plant resistance to pathogens. This assumption flowed from initial findings that these proteinsare commonly induced in resistant plants, expressing a hypersensitive necrotic response (HR) to pathogens ofviral, fungal and bacterial origin. PRs have been defined as 'proteins encoded by the host plant but inducedonly in pathological or related situations', the latter implying situations of non-pathogenic origin. In thisreview, brief information like biochemistry, source, regulation of gene expression, mode of action of defensemechanism of various pathogenesis-related proteins is investigated.

Keywords: HR, Pathogenesis-related Proteins, Plant defense, Systemic acquired resistance.

INTRODUCTION

The PR-proteins were defined as 'proteins coded by thehost plant but induced only in pathological or relatedsituations' (Antoniw et al., 1980). However, relatedproteins were identified that accumulate in normal(uninfected) plants in certain tissues or developmentalstages. These proteins are referred to as 'PR-like'proteins (Van Loon, 1999). In some situations, a genefor a PR-like protein expressed in a developmentallycontrolled manner may be inducible in some other planttissue in response to stresses. Thus, the distinctionbetween a PR-protein and a PR-like protein may be lessclear-cut in some situations. The PR-proteins areidentified easily in cell extracts of infected plants (VanLoon and Van Kammen, 1968, Gianinazzi and Vallee,1969). In fact, they are quite prominent in acid extractsof infected plants. Perhaps because of the need tofunction in a hostile environment, most PR-proteinsshow pH- and thermal stabilities and are quite resistantto proteolysis. In addition, the accumulation of asubclass of PR-proteins (typically the acidic forms) inthe apoplastic fluid (extracellular compartment) hassimplified the purification of several PR-proteins.Furthermore, the availability of several affinitychromatography procedures for the isolation of somePR-proteins (chitin and curdlan columns, for example)has been very useful in their rapid purification. Thus,many of the PR-proteins have been purified easily tohomogeneity and used for the preparation of antibodiesspecific for each group. The availability of antibodiesalso has led to the cloning of the corresponding cDNAsand genes for PR-proteins. Thus databases containseveral hundred sequences for PR-proteins from diverseplants. From the major cereals, cDNA or genomic

clones for a large number of PR-proteins have beenisolated Association of pathogenesis-related proteinswith induced resistanceThe notion that the enhanced resistance apparent uponchallenge inoculation depends on the same defensemechanisms as expressed after primary infection led tothe identification of common metabolic alterationsinduced systemically in response to local infection.Whereas induction of phytoalexins and cell wallrigidification are local reactions, accumulation ofpathogenesis-related proteins (PRs) extends into non-inoculated plant parts that, upon challenge, exhibitacquired resistance (Van Loon and Van Kammen,1970, Ryals et al., 1996). The proteins themselves arenot transported from the primary inoculated leaves, asdemonstrated elegantly by (Gianinazzi and Ahl, 1983)through the analysis of reciprocal grafts of Nicotianaspecies expressing electrophoretically differentproteins. While a link between PRs and acquiredresistance in virus-infected tobacco was immediatelyhypothesized (Van Loon and Van Kammen, 1970,Kassanis et al.,1974, Van Loon, 1975, Fraser, 1982)pointed out that PRs became apparent in non-inoculatedleaves distinctly later than acquired resistance appearedmanifest. However, in tissues already primed to expressPRs, challenge inoculation might lead to their earlierand faster accumulation. Moreover, a hybrid betweenN. glutinosa and N. debneyi constitutively expressedPRs and was highly resistant against TNV (Ahl andGianinazzi, 1982). Induction of PRs has since beenfound to be invariably linked to necrotizing infectionsgiving rise to SAR, and has been taken as a marker ofthe induced state (Ward et al.,1991, Uknes et al.,1992,Kessmann et al.,1994).

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Golshani, Fakheri, Behshad and Vashvaei 478

This notion has been reinforced by the characterizationin Arabidopsis of mutants that either are compromisedin both the production of PRs and the induction of SAR(npr1; Cao et al.,1994, nim1; Delaney et al.,1995), orare constitutive expressers of PR genes as well as SAR(cpr1; Bowling et al.,1994). PRs have been defined asplant proteins that are induced in pathological or relatedsituations (Van Loon et al.,1994). Although they areimplicated in plant defense, they have not beenidentified because of their anti-pathogenic action, butsolely because of their accumulation in infected plants.Eleven families of PRs have now been officiallyrecognized (Van Loon et al.,1994), but additionalpathogen-induced proteins with potential anti-pathogenic action keep being described (Broekaert etal.,1995). PRs have been identified in at least nine plantfamilies, with those in tobacco and tomatocharacterized best. Now, it is known that they comprisefour families of chitinases (PR-3, -4, -8 and -11), one ofβ-1,3-glucanases (PR-2), one of proteinase inhibitors(PR-6), and one specific peroxidase (PR-9), as well asthe PR-1 family with unknown biochemical properties,the thaumatin-like PR-5 family, and the birch allergenBetv1-related PR-10 family. Not all families arerepresented in any plant species, but each family maycomprise several members. Together the PRs form a setof pathogen-induced proteins that may be considered asstress proteins. In the past decades it has becomeevident that plants, when exposed to variousenvironmental stresses, respond by synthesizing sets ofspecific proteins. Well-known are the heat-shockproteins, that appear to be common to all livingorganisms, and are transiently induced when ambienttemperature exceeds some critical limit (Vierling,1991). Different sets of proteins are induced by e.g.drought stress or freezing temperatures. For instance,during cold acclimation hardy cultivars of alfalfasynthesize a number of proteins that supposedlyfunction in reducing the deleterious effects of lowtemperature on plant membranes. The plant hormoneabscisic acid (ABA) induces a partly similar set ofproteins and increases resistance to freezing stress,indicating that acclimation is hormone-controlled(Mohapatra et al.,1988, Heino et al., 1990). Similarproteins are induced by ABA during the acquisition ofdesiccation tolerance in developing seeds and upondrought stress of leaves (Skriver and Mundy, 1990).PRs may be considered as stress proteins produced inresponse to, particularly necrotizing, infections byviruses, viroids, fungi and bacteria, and thought tofunction in the acquired resistance against furtherinfection (Van Loon, 1989). However, in contrast tomost other types of stress proteins, they accumulate inplant tissues to levels that are easily detectable on gelsby general protein stains. Why these inducible PRs mayindividually reach up to 1% of the total soluble proteinin leaves, is unclear. Some of the PRs possess potential

antipathogenic activities (Linthorst, 1991, Van Loon etal., 1994).Chitinases, together with glucanases, could be directedagainst fungal cell walls and, perhaps, insects. Insectsare likely to be affected by proteinase inhibitors.Bacteria may be inhibited by the PR-8 family ofchitinases, which also possesses lysozyme activity. ThePR-9 peroxidase is of the lignin-forming type and couldbe involved in the strenghtening of cell walls. The PR-10 family has sequence similarity to ribonucleases andis the only family consisting of cytoplasmic proteins,but there is no evidence that PR-10 proteins are activeagainst e.g. viruses. The PR-1 and PR-5 proteins areoften strongly induced and seem to affect membranes,but their precise actions have not been elucidated. Theinducible PRs are mostly acidic proteins that aresecreted into the intercellular space of the leaf. Boththrough cDNA sequence homologies and on the basisof similar enzymatic activities, additional basiccounterparts have been identified. These basic PRsoccur at relatively low levels in the vacuole and,besides being induced upon infection, are expressed ina tissue-specific and developmentally-controlledmanner in leaves, roots and floral parts (Eyal and Fluhr,1991, Linthorst, 1991). Specific activities vary greatly,i.e. from 5 nKat/mg with laminar in as a substrate forthe inducible, acidic glucanase PR-2a to 23 and 1300nKat/mg for -2b and -2c, respectively, and 1100nKat/mg for the developmentally-controlled basicglucanase PR-2e (Kauffmann et al.,1987). It has beensuggested that in induced plants the accumulatedintercellular proteins form the first line of defense to achallenging pathogen and, if this fails and the tissue isdisrupted, the release of the vacuolar PRs functions as asecond line, engulfing the pathogen with lytic enzymes(Mauch and Staehelin, 1989). The constitutiveexpression of several of the basic proteins in olderleaves, roots and developing flowers could be similarlyconsidered as a protective mechanism against pathogeninvasion, possibly contributing to the often observedincrease in resistance with plant age. However, theorgan-specific expression of specific PR genes suggeststhat the proteins also play roles in normaldevelopmental processes.

A. Effects of pathogenesis-related proteins onexpression of resistanceConstitutive expression of individual PRs in transgenicplants can lead to reduced pathogen growth andsymptom expression, consistent with a role of PRs inthe expression of acquired resistance (Ryals etal.,1994). However, such effects are by no meansgeneral and pathogens may have evolved mechanismsto reduce the effects of PRs. Thus, many chitin-containing fungi are not inhibited by chitinases,presumably because the chitin in their cell walls isshielded by a protective layer. Such a layer may be lessdeveloped at growing hyphal tips, which can be lysed(Schlumbaum et al.,1986).


Significant suppression of disease symptoms caused bythe soil-borne fungus Rhizoctonia solani wasdemonstrated in tobacco or canola expressing avacuolar (class I) chitinase from bean (Broglie etal.,1991), the basic tobacco chitinase PR-3c (Lawton etal.,1993, Vierheilig et al.,1993), tobacco or cucumberPR-8 (Lawton et al.,1993), or the (class II) barleychitinase (Jach et al.,1995), but enhanced chitinaselevels caused no significant protection againstnicotianae (Neuhaus et al.,1991, Nielsen et al.,1993,Zhu et al.,1994) or Fusarium oxysporum (Van denElzen et al.,1993, Jongedijk et al.,1995). The reductionof R. solani in vacuole-targeted class I chitinasetransformed plants is fairly unexpected, becausetobacco roots constitutively express high levels of theirown class I chitinase but, nevertheless, are fullysusceptible. Antifungal activity of chitinases can besynergistically enhanced by β-1,3-glucanases, both invitro and in vivo. Thus, co-expression of chitinase andglucanase genes in tobacco enhanced resistance againstC. nicotianae (Zhu et al.,1994, Jach et al.,1995). Intomato, simultaneous expression of the basic tobaccochitinase PR-3d and glucanase PR-2e affordedsubstantial protection against F. oxysporum f. sp.lycopersici, whereas transgenic plants expressing eitherone of these genes were not protected (Jongedijk etal.,1995). Targeting the proteins to the apoplast was notmore effective, indicating that tonoplast leakagemustoccur sufficiently early to halt pathogen progress.Combinations of the acidic tobacco chitinase PR-3a andglucanase PR- 2b were not effective whenaccumulating either in the apoplast or in the vacuole,nor were combinations of an acidic glucanase with abasic chitinase, or vice versa. The situation appearslargely similar for the PR-1 and -5 proteins, which havebeen found to possess antifungal activity againstoomycetes, which lack chitin in their cell walls.Constitutive expression of PR-1a in tobacco reducedsymptoms caused by P. tabacina (Alexander etal.,1993, Lawton et al.,1993) and Phytophthoraparasitica f.sp. nicotianae (Alexander et al.,1993), butnot those provoked by the non-oomycete C. nicotianae,or the bacterial pathogen Pseudomonas syringae pv.Tabaci (Alexander et al.,1993). The various tomato andtobacco PR-1 proteins displayed inhibitory activity onthe growth of P. infestans in tomato leaf disc assays,with tomato PR-1c and tobacco PR- 1g being the mosteffective family members (Niderman et al.,1995). BasicPR-5 ('osmotin') likewise has antifungal activity againstP. infestans (Vigers et al.,1992, Zhu et al.,1996), but intransgenic tobacco no delay in symptoms caused by P.parasitica f.sp. nicotianae was apparent (Liu et al.,1994). So far, no results have been published onsuppression of pathogens in transgenic plantsexpressing PR-4, but basic tobacco PR-4c exhibitsantifungal activity in vitro against certain fungi andunder these conditions has been found to actsynergistically with basic tobacco PRs -2 and -3

(Ponstein et al.,1994). An additional 'SAR gene' intobacco, SAR 8.2. when expressed constitutively intransgenic tobacco, was also found to reduce diseasecaused by P. parasitica, but the protein has not beencharacterized (Lawton et al.,1993).Taken together,these observations do not indicate a significant role ofthe major, pathogen-inducible PRs in the enhancedresistance expressed upon challenge inoculation ofplants with SAR. Additional SAR genes, comprisingboth minor, developmentally-controlled PRs and thoseencoding mRNAs for which the protein has not beenidentified SAR 8.2 (Ward et al.,1991) glycine-richprotein (Van Kan et al., 1988, Linthorst, 1991) couldhave more effect. However, in as far as the activities ofPRs have been determined, these are directed onlyagainst fungi; PRs or similar proteins effective againstbacteria or viruses have, so far, not been identified. Arole of proteinase inhibitors against insect attack is wellestablished (Ryan, 1990), but SAR is not commonlyassociated with enhanced protection against insects.Screening specifically for antifungal, antibacterial andantiviral activities in plants has yielded PR-like proteinswith antifungal properties related to PR-families -4(Hejgaard et al., 1992) and -5 (Vigers et al.,1991), aswell as ribosome-inactivating proteins, thionins, lectinsand defensins (Linthorst, 1991). These proteinscommonly occur in storage organs, such as seeds andtubers, but may also be induced in leaves followingpathogen attack (Broekaert et al.,1995). Judging fromthe synergistic actions of some of these proteins whenexpressed together, it may be expected that whenmultiple SAR genes are coordinately expressed, such asin a HR, complementary actions of the resultingproteins could yield a strong anti-pathogenic potential.Moreover, when resistance responses are activatedupon challenge inoculation, PRs are induced again, andmore quickly and strongly than in non-induced plants.However, the apparent lack of PRs or other induceddefensive proteins with activity against bacteria andviruses is difficult to reconcile with the non-specificityof acquired resistance. It is not inconceivable that,besides the conspicuous PRs, several other compoundsare induced that have antipathogenic activities, but areyet to be discovered. The significance of the induciblePRs then becomes difficult to assess at present. It isintriguing that the thaumatin like PR-5 family isexpressed not only in response to pathogens, but alsoduring osmotic stress. Thus, osmotin (tobacco PR-5c)and its homolog NP24 (PR- 5a) in tomato wereindependently identified as being induced by salt stress(Singh et al., 1987) and infection (Stintzi et al.,1991).Interestingly, lipid transfer proteins can also be inducedby infection, have antifungal activity, and are expressedduring drought stress (Kader, 1997). Such observationssupport a role for PRs as stress proteins with functionsthat exceed their involvement in plant-pathogeninteractions.


(i) Pathogenesis-related (PR) protein 1.The first PR-1protein was discovered in 1970. Since then, a numberof PR-1 proteins have been identified in Arabidopsis,Hordeum vulgare (barley), Nicotiana tabacum(tobacco), Oryza sativa (rice), Piper longum (pepper),Solanum lycopersicum (tomato), Triticum sp. (wheat)and Zea mays (maize) (Liu and Xue, 2006). These PR-1having 14 to 17 kD molecular weight and mostly ofbasic nature. Non-expressors of Pathogenesis- RelatedGenes1 (NPR1) regulate systemic acquired resistancevia regulation pathogenesis related 1 (PR-1) inArabidopsis thaliana. The interaction of nucleus-localized NPR1 with TGA transcription factors, afterreduction of cysteine residues of NPR 1 by salicylicacid (SA) results in the activation of defense genes ofPR-1. In the absence of TAG 2 and/or SA expression ofPR-1 not occur in Arabidopsis thaliana (Després et al.,2000, Rochon et al.,2006). PR-1 proteins haveantifungal activity at the micromolar level against anumber of plant pathogenic fungi, including Uromycesfabae, Phytophthora infestans, and Erysiphe graminis(Niderman et al.,1995). The exact mode of action of theantifungal activities of these proteins are yet to beidentified but a PR-1-like protein, helothermine, fromthe Mexican banded lizard have been found to beinteracting with the membrane-channel proteins oftarget cells, inhibiting the release of Ca2+ (Monzingo etal.,1996).

(ii) â-Glucanase (PR2).Plant β -1,3-glucanases (ß-1,3-Gs)comprises of large and highly complex genefamiliesinvolved in pathogen defense as well as a wide range ofnormal developmental processes. β-1,3-Gs havemolecular mass in the range from 33 to 44 kDa (Hongand Meng, 2004, Saikia et al.,2005). These enzymeshave wide range of isoelectric pH. Most of the basicâ-1,3-Gs are localized in vacuoles of the plant cells whilethe acidic β -1,3-Gs are secreted outside the plant cell.Wounding, hormonal signals like methyl jasmonate andethylene (Wuand Bradford, 2003), pathogen attack likefungus Colletotrichum lagenarium (Ji and Ku, 2002)and some fungal elicitors releases from pathogen cellwall (Boller, 1995) can also induced â-1,3-Gs in thevarious parts of plant(Wu and Bradford, 2003, Saikia etal.,2005). The enzyme â-1,3-Gs was found to bestrongly induced by ultraviolet (UV-B; 280-320 nm)radiation in primary leaves of French bean (Phaseolusvulgaris), so that UV-induced DNA damage is aprimary step for the induction of â-1,3-Gs(Kucera etal.,2003). â-1,3- glucanases and chitinases are downregulated by combination of auxin and cytokinin whileAbscisic acid (ABA) at a concentration of 10 μMmarkedly inhibited the induction of â-1,3- glucanasesbut not of chitinases (Rezzonico, 1998, Wu et al.,2001).These enzymes are found in wide variety of plants likeArachis hypogaea (peanut), Cicer arietinum (chickpea),

Nicotiana tabacum (tobacco), etc. and having resistivityagainst various fungi like Aspergillus parasiticus, A.flavs, Blumeria graminis, Colletotrichum lagenarium,Fusarium culmorum, Fusarium oxysporum, fusariumudum, Macrophomina phaseolina and Treptomycessioyaensis (Rezzonico, 1998, Wu and Bradford, 2003,Hong and Meng, 2004, Wróbel-Kwiatkowska etal.,2004, Liang et al.,2005, Roy-Barman et al.,2006). â-1,3- glucanases are involves in hydrolytic cleavage ofthe 1,3-â-D-glucosidic linkages in â-1,3- glucans, amajor componant of fungi cell wall (Simmons, 1994,Høj and Fincher, 1995). So that cell lysis and cell deathoccur as a result of hydrolysis of glucans present in thecell wall of fungi.

(iii) Chitinases (PR3). Most of Chitinase havingmolecular mass in the range of 15 kDa and 43 kDa.Chitinase can be isolated from Cicer arietinum(chickpea) (Saikia et al., 2005), Cucumis sativus(cucumber), Hordeum vulgare (barley) (Kirubakaranand Sakthivel, 2006), Nicotiana tabacum (tobacco) (Puet al., 1996), Phaseolus vulgaris (black turtle bean)(Chu and Ng, 2005), Solanum lycopersicum (tomato)(Wu and Bradford, 2003) and Vitis vinifera (grapes)(Sluyter et al.,2005). Chitinases can be divided into twocategories: Exochitinases, demonstrating activity onlyfor the non-reducing end of the chitin chain; andEndochitinases, which hydrolyse internal â-1,4-glycoside bonds. Many plant endochitinases, especiallythose with a high isoelectric point, exhibit an additionallysozyme or lysozyme like activity (Collinge etal.,1993, Brunner et al.,1998, Schultze et al.,1998,Subroto et al.,1999). Chitinase and â-1,3-Glucanase aredifferentially regulated by Wounding, MethylJasmonate, Ethylene, and Gibberellin. Wounding andmethyl jasmonate induces gene chi 9 for Chitinasesexpression in the tomato seeds (Wu and Bradford,2003). In some study, it is also found that Chitinasegene are also expressed in response to stress like coldup to -2 to -5ºC (Yeh et al., 2000). These Chitinaseshave significant antifungal activities against plantpathogenic fungi like Alternaria sp. For graindiscoloration of rice, Bipolaris oryzae for brown spot ofrice, Botrytis cinerea for blight of Tobacco, Curvularialunata for leaf spot of clover, Fusarium oxysporum, F.udum, Mycosphaerella arachidicola, Pestalotia theaefor leaf spot of tea and Rhizoctonia solani for sheathblight of rice (Chu and Ng 2005, Saikia et al.,2005,Kirubakaran and Sakthivel, 2006). The main substrateof Chitinases is chitin - a natural homopolymer of â-1,4- inked N-acetylglucosamine residues (Kasprzewska,2003). The mode of action of PR-3 proteins is relativelysimple i.e. Chitinases cleaves the cell wall chitinpolymers in situ, resulting in a weakened cell wall andrendering fungal cells osmotically sensitive (Jach etal.,1995).


(iv) Chitin Binding Protein (CBP, PR4). All chitinbinding proteins do not possess antifungal activities.CBP can be isolate from plant Beta vulgaris (sugarbeat), Hydrangea macrophylla (hortensia), Nicotianatabacum (tobacco), Piper longum (pepper), Solanumlycopersicum (tomato) and Solanum tuberosum (potato)and bacteria like Streptomyces tendae (Nielsen etal.,1997, Bormann et al.,1999, Lee et al., 2001,Yangand Gong, 2002). Moleculer weight of the CBPwas found to be in the range of 9 kDa to30 kDa andhaving basic isoelectric pH (Nielsen et al.,1997,Bormann et al.,1999,Yang and Gong, 2002).Expression of theCACBP1 chitin-binding proteinisolated from cDNA library of pepper (Capsicumannuum L.) (CACBP1) gene was rapidly induced in theincompatible interactions upon pathogen infection,ethephon, methyl jasmonate or wounding (experimentalmodel plant pepper). The CACBP1 gene was organ-specifically regulated in plants. High level ofexpression occurs in phloem of vascular bundles inleaves of pepper (Lee et al.,2001, Wan et al.,2008).CBPshows strong inhibitory effect against fungi Aspergillusspecies, Cercosporabeticola, Xanthomonas campestrisand manymore and several crop fungalpathogen(Nielsen et al.,1997, Bormann et al.,1999,Leeet al.,2001, Yang and Gong, 2002). Enzymetically CBPhas not any function but itbinds to insoluble chitin andenhances hydrolysis of chitin by other enzyme likeChitinase (Houston et al.,2005, Vaaje-Kolstad etal.,2005).

(v)Thaumatin-Like Protein (TLP, PR5). Thaumatin-like proteins comprise of polypeptides classes that sharehomology with thaumatin, sweet protein fromThaumatococcus danielli (Bennett) Benth (Cornelissenet al.,1986). Thaumatin-like proteins can be isolatedfrom Hordeum vulgare (barley), Actinidia deliciosa(kiwifruit), Zea mays (maize), Pseudotsuga menziesii(douglas-firs), Nicotiana tabacum (tobacco), Solanumlycopersicum (tomato) and Triticum sp. (wheat)(Wurms et al.,1999, Fecht-Christoffers et al.,2003,Anand et al.,2004, Zamani et al., 2004). Most of theTLPs have a molecular weight in the range of 18 kDa to25 kDa and have a pH in the range from 4.5 to 5.5(Fecht-Christoffers et al.,2003, Zamani et al.,2004).Constitutive levels of Thumatin-Like Protein istypically absent in healthy plants, with the proteinsbeing induced exclusively in response to wounding orto pathogen attack like Uncinula necator, Phomopsisviticola (Monteiro et al.,2003). Although the specificfunction of many PR5 in plants is unknown, they areinvolved in the Acquired Systemic Resistance and inresponse to biotic stress, causing the inhibition ofhyphal growth and reduction of spore germination,probably by a membrane permeabilization mechanismand/or by interaction with pathogen receptors(Thompson et al., 2007). Linusitin is a 25-kDaThaumatin-like Protein isolated from flax seeds.Linustin shows antifungal activity against Alternariaalternata by the mechanism of membranepermeabilization. Concentration of protein and lipid

and composition of cell wall of fungi play a major rolein these mechanisms (Anzlovar et al.,1998). In onestudy by (Menu-Bouaouiche et al.,2003), Thaumatin-like proteins were isolated from cherry, apple andbanana shows antifungal activity against Verticilliumalboatrum and having endo- â1,3-glucanase activity.

(vi) Proteinase inhibitors (PR-6). The peptidesbelonging to the PR-6 family are defined as a subclassof serine proteinase inhibitors (PIs) related to the''tomato/ potato inhibitor I'' (Glazebrook, 2005) andhave a typical molecular size of _8 kDa (Van Loonetal., 1994) The larger group of serine PIs, which are thebest studied PIs, are sub-divided, next to this PR-6family, in tomato/potato class II PIs, Bowman-Birk PIsand Kunitz-type PIs, reviewed in (Datta andMuthukrishnan, 1999, Haq et al., 2004). This proposedoriginal definition and classification of the PR-6 familyis debatable, since it is limited to a sole sub-class ofserine PIs, while all types of PIs can interact withproteinases from plant-attacking organisms and couldhave a role in plant defense (reviewed in (Christellerand Laing, 2005, Haq et al.,2004). Therefore, in theliterature the general term PI is often used instead ofPR-6. This section also presents some examples ofstudies concerning PIs, which are not all strictlyclassified as PR-6-type proteins. PIs have the propertyto bind proteinases and control proteinase activity, ageneral function involved in many biochemicalprocesses, and therefore could have multiple functionsin planta e.g. the regulation of endogenous proteinasesduring seed dormancy, reserve protein mobilization;reviewed in (Haq et al., 2004), apart from theirproposed defensive role reviewed in (Christeller andLaing, 2005, Haq et al.,2004). Concerning their role indefense, PIs may act by reducing the ability of theattacker to (i) use its lytic enzymes necessary forpathogenicity for fungi (Dunaevskii, et al.,2005), (ii)complete its replication cycles for viruses (Gutierrez-Campos et al.,1999), or (iii) obtain nutrition throughdigestion of host proteins and thereby limit the releasedamount of amino acids for nematodes, insects (Urwin etal.,1997, Vila et al.,2005).Different studies havereported that PI genes can be induced upon inoculationwith micro-organisms. For instance in tomato,induction of PI genes was shown upon inoculation withPhythophtora infestans (Peng and Black, 1976) andPseudomonas syringae pv. Tomato (Pautot et al.,1991).(Terras et al.,1993) showed an in vitro antimicrobialeffect, albeit relatively weak, of barley trypsininhibitors to four important fungal plant pathogensincluding Alternaria brassicicola, Ascochyta pisi,Fusarium culmorum and Verticillium dahliae.Interestingly, this activity increased synergistically incombination with other PR proteins, more specificallythionins (PR-13) (Terras et al.,1993). As the role ofspecific microbial proteinases in microbialpathogenicity is not always clear, the effect of plant PIson the activity of these enzymes has not been studiedintensively.


Yet, (Dunaevskii et al., 2005) showed that buckwheattrypsin inhibitors were able to inhibit in vitroproteinases of B. cinerea, likely to be necessary forpathogenesis (Ten Have et al., 2004). Also, (Lorito etal.,1994) suggested that PIs could block the synthesis ofchitin in fungal cell walls and consequently fungalgrowth, through inhibition of endogenous trypsinnecessary for active chitin synthase (Machida and Saito,1993). The in vivo effect of PIs on defense againstmicrobial pathogens was also shown as there are reportsof enhanced resistance in plants overexpressing PIgenes. For instance, heterologous overexpression ofNicotiana alata PI genes in transgenic tobacco resultedin enhanced resistance against B. cinerea(Charity etal.,2005). Recently, it was shown that endogenousoverexpression of two A. thaliana PI genes, includingone PR-6-type gene, in transgenic A. thaliana resultedin enhanced resistance against B. cinerea (Chassot etal.,2007). Nevertheless, hitherto most of the studies onPIs have focused on their role in plant-insectinteractions. It was found that PI genes are inducedupon insect attack and mechanical wounding (Corderoet al., 1994, Glazebrook, 2005). The wounding, leadingto activation of PI genes, is believed to mimic thechewing action of herbivorous insects. These insect andwound-induced responses have been extensivelystudied, especially the wound-activated systeminsignaling pathway in tomato (Constabel et al., 1995),and are generally linked to the octadecanoid pathwayand JA signaling networks. In A. thaliana, insect attackor wounding activates JA and ET signaling pathways(De Vos et al., 2005) giving rise to systemic inducedresistance (De Vos et al.,2005). However, general bothwounding and microbial pathogen attack activate JAsignaling pathways in A. thaliana. Hereby, theAtMYC2 and the ethylene response (ERFs)transcription factors play essential roles to discriminatebetween either wounding- or microbial pathogen-related JA responses and to regulate the activation ofthe appropriate set of genes (Anderson et al., 2004,Lorenzo et al.,2004). More specifically, while AtMYC2transcription factors activate wound responsive genes(i.e. PI genes), ERFs activate pathogen-responsivegenes (i.e. the PDF gene AtPDF1.2a) (Lorenzo etal.,2004). Recently it could also be demonstrated thatinsects try to reduce wound-induced expression of PIgenes in the plant, i.e. by components in theirregurgitants (Lawrence et al., 2007) or, by making useof a decoy mechanism which activates antagonizingsignal pathways in the plant (Zarate et al.,2007). Withrespect to their anti-insect activity, PIs could act againstthe digestive proteases (e.g. trypsin, chymotrypsin)used by herbivorous insects. This was demonstrated inseveral in vitro experiments by a reduced growth rate ofinsects fed on artificial diets containing PIs compared tothe growth rate of insects fed on diets without PIs(Harsulkar et al., 1999, Markwick et al.,1995, Tamhaneet al., 2005).

The effect of PIs on insect attack in vivo was alsoshown as there are several reports of transgenic plants,expressing a heterologous PI gene and making themmore resistant to insect attack reviewed in (Haq etal.,2004)). For instance, it was shown thatoverexpression of a PI gene from Nicotiana alata intransgenic apple plants inhibits the normal developmentof light-brown apple moth, Epiphyas postvittana(Maheswaran et al.,2007). (Duan et al.,1996) showedthat transgenic rice plants, harboring an introducedpotato PI gene, were more resistant to the pink stemborer (Sesamia inferens). (Vila et al., 2005)demonstrated that expression of a maize PI gene in riceplants enhanced resistance against the striped stemborer (Chilo suppressalis). No reports were found onincreased insect resistance upon overexpression of a PIgene in the plant from which they originated.

B. Characterization of plant PR-10 proteinsMore than 100 PR-10 or PR-10-related sequences havebeen identified from various flowering plants. PR-10proteins are identified primarily as IPR proteins,including tree pollen allergens and major food allergens(Wen et al.,1997). They have been reported in a varietyof higher plant species of both angiosperms andgymnosperms. Among them, PR-10 proteins have beenfound in numerous dicots, including parsley(Petroselinum crispum) (Somssich et al.,1986) pea(Pisum sativum) (Fristensky et al.,1988) potato(Solanum tuberosum) (Matton and Brisson, 1989),white birch (Betula verrucosa) (Breiteneder et al.,1989,Swoboda et al.,1995), bean (Phaseolus vulgaris L.)(Walter et al., 1990), soybean (Glycine max cvMandarin) (Crowell et al.,1992), alder (Alnusglutinosa)(Breiteneder et al.,1992), apple (Malus domestica)(Vanek-Krebitz et al.,1995), celery (Apiumgr aveolens)(Breiteneder et al.,1995), and alfalfa (Medicago sativa)(Bredaet al.,1996, Esnault et al., 1993). Among themonocots, PR-10 proteins are known to occur inasparagus (Asparagus officinalis)(Warneret al.,1992),rice (Oryza sativa)(Midoh and Iwata, 1996), lily(Lilium longiflorum) (Huanget al.,1997), and sorghum(Sorghum bicolor) (Loet al.,1999). Amonggymnosperms, PR-10 proteins have been found in sugarpine (Pinus lambertiana), eastern white pine (Pinusstrobus), western white pine (Pinus monticola)(Ekramoddoullah et al.,1995, Yuet al.,2000), Douglas-fir (Pseudotsuga menziesii) (Ekramoddoullah etal.,2000), maritime pine (Pinus pinaster) (Dubosetal.,2001), and white spruce (Picea glauca) (Mattheus etal.,2003).

C. Induced expression by biotic and abiotic stressesMost genes of the PR 10 IPR group have an openreading frame (ORF) from 456 to 489 bp encoding apolypeptide of 151-162 amino acids with molecularmasses of 15-18 kDa. The inducible expression of PR-10 genes in response to attacks by pathogens has beenwidely investigated in a number of plant species.


Pathogens triggering a PR-10 response include viruses(Pu¨ hringer et al.,2000, Park et al.,2004, Pinto andRicardo, 1995), bacteria(Breda et al.,1996, Esnault etal.,1993, Robert et al.,2001), and fungus (Swoboda etal.,1995, Walter et al.,1990, Liu et al.,2005, Liu etal.,2003). Immunocyto chemical localization has shownthat the PR-10 proteins bind to the cell wall of blisterrust fungus (Cronartium ribicola) both intracellularlyand extracellularly in infected sugar pine needles(Ekramoddoullah, 2004). Immunofluorescence analysishas revealed that a Douglas-fir PR-10 protein is inducedin cortical tissues of roots infected with the fungalpathogen, Phellinus sulphurascens Pilat. GUS activityregulated by the Ypr10*a promoter from apple (Malusdomestica) is induced in young leaves by multiplestress factors, including pathogen attack and a fungalelicitor (Pu¨ hringer et al.,2000).Wound treatmentusually induces PR-10 expression. As quantified byNorthern blot and PR-10 promoterb- glucuronidasefusion analysis, an asparagus PR-10 gene (AoPR1)transcript is accumulated after wounding(Warner etal.,1992, Warner et al.,1994). Potato PR-10a is rapidlyinduced by wounding or by an elicitor, and becomesdetectable 6 h after treatment, but the accumulation ofPR-10c in potato is unaffected by wounding or by anelicitor (Constabel and Brisson, 1995, Despre´s etal.,1995). Differential expression profiles have beenreported for birch PR-10a and PR-10c in response uponwounding (Poupard et al.,1998). In western white pine,both PR-10 mRNA transcripts and proteins are rapidlyinduced upon wounding(Liu et al.,2005, Liu etal.,2003). Furthermore, wounding can mimicCronartium ribicola disease-activated PR-10accumulation pattern in western white pine needles.Cronartium ribicola infection leads to expression of 10PR-10 isoforms in needles, while nine of themaccumulate after wounding(Liu et al.,2003).As animportant environmental factor, cold hardiness affectsthe PR-10 expression. PR-10 protein accumulates to thehighest level in the roots of sugar pine and westernwhite pine in winter(Ekramoddoullah et al.,1995). Theaccumulation of PR-10 proteins by cold acclimation hasalso been observed in peach (Wisniewskiet al.,2004)and mulberry (Ukajiet al., 2004).The expression of PR-10 genes is also induced by other abiotic stresses, forexample, by salinity in the roots of rice (Moonsetal.,1997), by drought stress in maritime pine(Dubos etal.,2001) and hot pepper(Park et al.,2004), bydormancy in a desert legume (Retama raetam) (Pnueliet al., 2002), by copper stress and other relatedoxidative stress in roots and leaves of birch (Betulapendula) (Koistinen et al.,2002, Utriainen et al.,1998),and by ultraviolet radiation in Lupinus albus L. (Pintoand Ricardo,1995).PR-10 gene expression is alsoregulated by plant hormones and defense-relatedsignaling molecules, including jasmonic acid(Liu etal.,2003, McGee et al., 2001, Moons et al.,1997,Rakwal et al.,2001, Wang et al.,1999), abscisic acid(ABA) (Wang et al., 1999), and salicylic acid (McGeeet al.,2001). Okadaic acid, a specific inhibitor of type 1

or type 2A serine/threonine protein phosphatase,enhances wound-induced PR-10 protein synthesis (Liuet al.,2003). These data suggest that defense-relatedsignal chemicals and protein phosphorylation areinvolved in the signal transduction pathway leading toPR-10 activation. Based on investigation using lilyanthers (Wang et al.,1999), have proposed that twoseparate signal transduction pathways are involved inthe induction of PR-10 genes by ABA and methaljasmonate (MeJA), respectively.

D. Plant defensins (PR-12)In 1990 the first plant defenses, isolated from wheat(Colilla et al,1990) and barley(Mendez et al, 1990),were originally categorized as a novel type thionin (seefurther) because of their similar molecular size (5 kDa)and similar number of cysteines (Boyes et al.,2001).However, as the structure (including the positions of thedisulfide bonds)was not related to known and bthionins(Bruixet al,1993), they were grouped apart as g-thionins. Later, because of their antimicrobial propertiesand their structural similarity to mammalian and insectdefensins, g-thionins were renamed ''plant defensins''(Terraset al,1995). PDFs were taken up in the group ofPR proteins and classified as the PR-12 family whenTerras et al. (Terras et al.,1995) discovered twoantifungal radish (Raphanus sativus) defensins (Rs-AFP3, Rs-AFP4) that were barely detectable in healthyuninfected leaves but accumulated at high levels afterfungal infection. Hitherto, numerous PDFs could bepurified from several plant species and plant tissuessuch as seeds, stems, roots, leaves and floral organs andshow a wide range of in vitro biological activitiesincluding a-amylase activity, ion-channel blocking andantibacterial activity (reviewed by Lay and Anderson,2005, Pellegrini and Franco, 2005, Thomma etal.,2002). However, their best characterized activity isthe ability to inhibit the growth of a broad range offilamentous fungi and yeasts in vitro. For instance,(Terraset al,1993, Terras et al., 1995)described a broadspectrum antifungal activity of several plant defensinsisolated from Brassicaceae species, including radish,against several filamentous fungi including B. cinerea,A. brassicicola and F. culmorum. The presence ofinorganic salts and especially divalent cations generallyantagonized the antifungal activity of plant defensins,although the cation sensitivity varied with the defensingand the fungus used (Terras et al.,1993). Recently,insight was gained in the mode of antifungal action oftwo different plant defensins from radish and dahlia(Dahlia merckii), Rs-AFP2 and Dm-AMP1,respectively (Thevissen et al.,2000, Thevissen etal.,2004). Both peptides bound on distinct sphingolipids(glucosylceramide and manosyldiinositolphosphorylceramide forRs-AFP2 and Dm-AMP1,respectively) in fungal membranes and, consequently,showed a different specificity against fungal and yeastsspecies, including the human pathogen Candidaalbicans (Thevissen et al.,2004).


The precise in vivo function of PDFs remains unclearand different roles have been attributed to them. ManyPDFs are expressed abundantly e.g. in seed (Terras etal,1995) whereas others are developmentally regulated(Vanoosthuyse et al,2001) or induced by differentabiotic and biotic stress factors, including cold (Koikeet al.,2002), drought (Do et al.,2004), heavy metals(Mirouze et al.,2006), potassium starvation(Armengaudet al.,2004), or microbial pathogens (Penninckx etal,1996, Terras et al,1995, Zimmerli et al, 2004). Thelatter, in addition to their in vitro antimicrobial activity,indicates a role of PDFs in the plant defense response,which is further emphasized by enhanced diseaseresistance phenotypes observed in different plantspecies heterologously overexpressing PDFgenes. Forinstance, (Terras et al.,1995) showed that transgenictobacco overexpressing the Rs-AFP2 gene was moreresistant to the fungal pathogen Alternaria longipes.Another study described enhanced resistance oftransgenic potato to Verticillium dahliaebyoverexpression of an alfalfa PDF gene (Gaoet al.,2000).So far, there are no reports of enhanced resistance byoverexpression of a PDF gene in the plant from which itoriginated.

E. Thionins (PR-13)Like PDFs, thionins are small (5 kDa), usually basic,cysteine-rich peptides and were originally isolated fromcereals (Bohlmannet al.,1988). Hitherto, around 100

thionin gene sequences have been identified in 15different plant species reviewed by(Stec, 2006).Induced expression of leaf thionins could be shownupon fungal infection in barley and A. thaliana(Bohlmann et al., 1988, Epple et al,1995) andconsequently they were classified as the PR-13 familyof PR proteins.The main characteristic of thionins is their broad invitro antifungal and antibacterial activity (Bohlmann etal.,1988, Cammue et al.,1992)and like plant defensins,their antimicrobial effects lead to the permeabilizationof cell membranes (Stec, 2006, Thevissen et al,1996).Antimicrobial activity was demonstrated againstphytopathogenic bacteria see references in(Castro andFontes, 2005), phytopathogenic fungi (Cammue etal.,1992, Terras et al,1993), yeast (Castro and Fontes,2005), and mammalian cell lines(Carrasco et al,1981).Combination of thionins and LTPs resulted insynergistically antifungal activity, suggesting theseproteins may cooperate in membrane binding and/orpermeabilization (Molina et al,1993), (Stec,2006).There are also several reports on transgenicplants expressing a thionin gene and making them moreresistant to fungal or bacterial pathogen attack. Forexample (Chan et al.,2005) showed that heterologousoverexpression of the A. thaliana Thi2.1gene intransgenic tomato made these plants more resistant tobacterial wilt and Fusarium wilt.

Table 1: Main properties of classified families of PR proteins.

Family Type member Typical size(kDa)

Properties Proposedmicrobial target

Original reference

PR-1 Tobacco PR-1a 15 Antifungal Unknown (Antoniwetal,1980)

PR-2 Tobacco PR-2 30 b-1,3-Glucanase b-1,3-Glucan (Antoniw et al,1980)

PR-3 Tobacco P, Q 25-30 Chitinase (class I,II,IV,V,VI,VI)

Chitin (Van Loon,1982)

PR-4 Tobacco ‘R’ 15-20 Chitinase class I,II Chitin (Van Loon, 1982)PR-5 Tobacco S 25 Thaumatin-like Membrane (Van Loon, 1982)PR-6 Tomato Inhibitor I 8 Proteinase-inhibitor –a (Green and

Ryan,1972)PR-7 Tomato P69 75 Endoproteinase –a (Vera and

Conejero,1988)PR-8 Cucumber chitinase 28 Chitinase class III Chitin (Me´ trauxet

al,1988)PR-9 Tobacco‘lignin-

formingperoxidase’35 Peroxidase –a (Lagriminiet

al,1987)PR-10 Parsley ‘PR1’ 17 ‘Ribonuclease-like’ –a (Somssichet

al,1986)PR-11 Tobacco ‘class V’

chitinase40 Chitinase class I Chitin (Melcherset

al,1994)PR-12 Radish Rs-AFP3 5 Defensin Membrane (Terraset al,1995)PR-13 ArabidopsisTHI2.1 5 Thionin Membrane (Eppleet al,1995)PR-14 Barley LTP4 9 Lipid-transfer protein Membrane (Garcıa-Olmedoet

al,1995)PR-15 Barley OxOa (germin) 20 Oxalate oxidase –a (Zhanget al,1995)PR-16 Barley OxOLP 20 ‘Oxalate oxidase-like’ –a (Weiet al,1998)PR-17 Tobacco PRp27 27 Unknown –a (Okushimaet al,

2000)

Table was adjusted from http://www.bio.uu.nl/wfytopath/PR-families.htmaNo in vitro antimicrobial activity reported.

http://www.bio.uu.nl/wfytopath/PR-families.htm


Also, high-level expression of a hordothionin gene frombarley in transgenic tobacco conferred resistance toPseudomonas syringae (Carmona et al,1993) andenhanced resistance to bacterial diseases was describedin transgenic rice plants overproducing an oat cell wall-bound thionin (Iwai et al., 2002). Interestingly, (Eppleet al,1997) described that over expression of theendogenous Thi2.1thionin gene enhanced resistance ofA. thaliana against Fusarium oxysporum. The latterstudy reports on homologous over expression andproves in this way a direct role in defense for thisthionin gene in A. thaliana. No studies on transgenicplants with RNAi or knockout of thionin genes andtheir possible implication on plant defense were found.Apart from their putative defensive function based uponthese data, other in vivo functions have been proposedfor thionins. There are indications for a regulatory roleas thionins have thioredoxin activity and hereby couldact as secondary messengers in the redox regulation onenzymes (Johnson et al,1987). Thionins found in seeds,could also function as storage proteins, especially as asource of sulfur (Castro and Fontes, 2005).To be complete, the main properties of all the hithertoclassified PR proteins are summarized in Table 1.

F. Lipid transfer proteins (PR-14)Lipid transfer proteins (LTPs) are small, cationic,cysteine-rich peptides found in various plant speciesreviews in(Carvalho and Gomes, 2001,Kader, 1996,Kader, 1997, Yeats and Rose, 2008), includingbarley(Molinaet al,1993), grapevine(Giraultet al.,2008),wheat(Sunet al.,2008), A. thaliana and spinach(Seguraet al,1993)and onion(Cammueet al,1995). PlantLTPs are sub-divided into two families, LTP1s andLTP2s, which present molecular masses of around 9and 7 kDa, respectively. Apart from these differences inmolecular size, the other characteristics of members ofthese two families, such as high pI and the pattern offour conserved disulfide bridges, are similar (Carvalhoand Gomes, 2007). LTPs were named because of theirability to facilitate the transfer of phospholipidsbetween membranes in vitro(Kader,1975). They areable to transfer various types of lipids includingphosphatidylinositol, phosphatidylcholine andgalactolipids (Castro and Fontes, 2005). Due to this lowspecificity for the lipid substrate, plant LTPs are alsonamed ''non-specific lipid transfer proteins''(Kader,1996). As LTP gene expressionwas also foundto respond to infection with pathogens (García-Olmedoet al,1995) they were classified as PR proteins. LTPspossess a signal peptide, targeting them to the cellsecretory pathway (Carvalho and Gomes, 2007).Various LTPs have been shown to be localized at thecell wall, as was demonstrated for the A. thalianaLTP1gene product (Thoma et al.,1993). This extra-cellular location is not a general rule as LTPs have alsobeen found in glyoxosomes where their activity waslinked to lipid catabolism (Tsuboi et al,1992) and in

protein storage vacuoles of seeds (Carvalho etal.,2001).LTPgenes are generally expressed in leaves and inflowers, and rarely in roots (Arondel et al.,2000). Inleaves, LTPgenes are usually expressed at highlevels inyoung expanding leaves and hereby LTPs weresuggested to play a role in the transport of monomers ofcutin and found to be associated with cutin and waxassembly (Chassot et al., 2007, Pyee et al.,1994).Though, the majority of LTPgenes seem to beexpressed in flowers or flower organs (Arondel et al,2000, Park et al.,2000, Pyee et al., 1994). (Park et al.,2000) showed that an LTP is necessary for pollenadherence to the stigma during pollen elongation in theLilium longiflorum. Next to their inducibility uponpathogen infection (García-Olmedo et al.,1995, Jung etal., 2003), LTPgenes are also responsive to abioticstresses like drought, cold and salt (Jang et al.,2004,Jung et al., 2003). Based on these findings inexpression patterns, many biological activities havebeen suggested for LTPs (Yeats and Rose, 2008),including roles in cutin synthesis (Penninckx etal.,1998), b-oxidation (Tsuboi et al.,1992), plantdefense signaling (Buhot et al., 2001, Maldonado etal.,2002), and plant defense (Girault et al.,2008,Kristensen et al., 2000, Molina et al., 1993).Concerning their role in plant defense, various LTPshave been shown to have in vitro antimicrobialactivities against fungi and bacteria (Cammue etal.,1995, Molina et al., 1993,Wang et al., 2004). Thisobserved antimicrobial activity could result from theinteraction of LTPs with biological membranes,possibly leading to membrane permeabilization (Kader,1996). There are also several reports on transgenicoverexpression of LTP genes resulting in enhancedtolerance to pathogen infection. For instance, transgenictobacco expressing a barley LTP gene showedenhanced resistance against Pseudomonas syringae pv.Tabaci (Molina and Garcia-Olmedo,1997). TransgenicA. thaliana, overexpressing a barley LTP gene, showedenhanced resistance against Pseudomonas syringaepv.tomato and B. cinerea (Junget al., 2005). Concerninghomologous overexpression experiments, (Chassot etal., 2007) described that endogenous overexpression ofthree LTP-like genes in A. thaliana resulted inenhanced tolerance to B. cinerea.

CONCLUSIONS

PR proteins play important role in disease resistance,seed germination and also help the plant to adapt to theenvironmental stress. The increasing knowledge aboutthe PR proteins gives better idea regarding thedevelopment and defense system of plants. Primaryaspects of the gene regulation of the PR proteins areunderstood but the study of exact mechanism of generegulation and receptor cascade will open new ways forthe plant genetic engineering technology for cropimprovement.


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